1. Field of the Invention
This invention is in the field of medicinal chemistry. In particular, the invention relates to substituted 4H-chromenes and analogs, and the discovery that these compounds are activators of caspases and inducers of apoptosis.
The invention also relates to the use of these compounds as therapeutically effective anti-cancer agents.
2. Related Art
Organisms eliminate unwanted cells by a process variously known as regulated cell death, programmed cell death or apoptosis. Such cell death occurs as a normal aspect of animal development, as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59–86 (1951); Glucksmann, A., Archives de Biologie 76:419–437 (1965); Ellis, et al., Dev. 112:591–603 (1991); Vaux, et al., Cell 76:777–779 (1994)). Apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells and eliminates cells that have already performed their function. Additionally, apoptosis occurs in response to various physiological stresses, such as hypoxia or ischemia (PCT published application WO96/20721).
There are a number of morphological changes shared by cells experiencing regulated cell death, including plasma and nuclear membrane blebbing, cell shrinkage (condensation of nucleoplasm and cytoplasm), organelle relocalization and compaction, chromatin condensation and production of apoptotic bodies (membrane enclosed particles containing intracellular material) (Orrenius, S., J. Internal Medicine 237:529–536 (1995)).
Apoptosis is achieved through an endogenous mechanism of cellular suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9–34). A cell activates its internally encoded suicide program as a result of either internal or external signals. The suicide program is executed through the activation of a carefully regulated genetic program (Wyllie, et al., Int. Rev. Cyt. 68.251 (1980); Ellis, et al., Ann. Rev. Cell Bio. 7:663 (1991)). Apoptotic cells and bodies are usually recognized and cleared by neighboring cells or macrophages before lysis. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells (Orrenius, S., J. Internal Medicine 237:529–536 (1995)).
It has been found that a group of proteases are a key element in apoptosis (see, e.g., Thornberry, Chemistry and Biology 5:R97–R103 (1998); Thornberry, British Med. Bull. 53:478–490 (1996)). Genetic studies in the nematode Caenorhabditis elegans revealed that apoptotic cell death involves at least 14 genes, 2 of which are the pro-apoptotic (death-promoting) ced (for cell death abnormal) genes, ced-3 and ced-4. CED-3 is homologous to interleukin 1 beta-converting enzyme, a cysteine protease, which is now called caspase-1. When these data were ultimately applied to mammals, and upon further extensive investigation, it was found that the mammalian apoptosis system appears to involve a cascade of caspases, or a system that behaves like a cascade of caspases. At present, the caspase family of cysteine proteases comprises 14 different members, and more may be discovered in the future. All known caspases are synthesized as zymogens that require cleavage at an aspartyl residue prior to forming the active enzyme. Thus, caspases are capable of activating other caspases, in the manner of an amplifying cascade.
Apoptosis and caspases are thought to be crucial in the development of cancer (Apoptosis and Cancer Chemotherapy, Hickman and Dive, eds., Humana Press (1999)). There is mounting evidence that cancer cells, while containing caspases, lack parts of the molecular machinery that activates the caspase cascade. This makes the cancer cells lose their capacity to undergo cellular suicide and the cells become immortal and cancerous. In the case of the apoptosis process, control points are known to exist that represent points for intervention leading to activation. These control points include the CED-9-BCL-like and CED-3-ICE-like gene family products, which are intrinsic proteins regulating the decision of a cell to survive or die and executing part of the cell death process itself, respectively (see, Schmitt, et al., Biochem. Cell. Biol. 75:301–314 (1997)). BCL-like proteins include BCL-xL and BAX-alpha, which appear to function upstream of caspase activation. BCL-xL appears to prevent activation of the apoptotic protease cascade, whereas BAX-alpha accelerates activation of the apoptotic protease cascade.
It has been shown that chemotherapeutic (anti-cancer) drugs can trigger cancer cells to undergo suicide by activating the dormant caspase cascade. This may be a crucial aspect of the mode of action of most, if not all, known anticancer drugs (Los, et al., Blood 90:3118–3129 (1997); Friesen, et al., Nat. Med. 2:574 (1996)). The mechanism of action of current antineoplastic drugs frequently involves an attack at specific phases of the cell cycle. In brief, the cell cycle refers to the stages through which cells normally progress during their lifetime. Normally, cells exist in a resting phase termed Go. During multiplication, cells progress to a stage in which DNA synthesis occurs, termed S. Later, cell division, or mitosis, occurs in a phase called M. Antineoplastic drugs, such as cytosine arabinoside, hydroxyurea, 6-mercaptopurine, and methotrexate are S phase specific, whereas antineoplastic drugs, such as vincristine, vinblastine, and paclitaxel are M phase specific. Many slow growing tumors, e.g., colon cancers, exist primarily in the Go phase, whereas rapidly proliferating normal tissues, e.g., bone marrow, exist primarily in the S or M phase. Thus, a drug like 6-mercaptopurine can cause bone marrow toxicity while remaining ineffective for a slow growing tumor. Further aspects of the chemotherapy of neoplastic diseases are known to those skilled in the art (see, e.g., Hardman, et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, New York (1996), pp. 1225–1287). Thus, it is clear that the possibility exists for the activation of the caspase cascade, although the exact mechanisms for doing so are not clear at this point. It is equally clear that insufficient activity of the caspase cascade and consequent apoptotic events are implicated in various types of cancer. The development of caspase cascade activators and inducers of apoptosis is a highly desirable goal in the development of therapeutically effective antineoplastic agents. Moreover, since autoimmune disease and certain degenerative diseases also involve the proliferation of abnormal cells, therapeutic treatment for these diseases could also involve the enhancement of the apoptotic process through the administration of appropriate caspase cascade activators and inducers of apoptosis.
EP537949 discloses derivatives of 4H-naphthol[1,2-b]pyran as antiproliferatives:
wherein,    each R1 is independently halo, trifluoromethyl, C1-4 alkoxy, hydroxy, nitro, C1-4 alkyl, C1-4 alkylthio, hydroxy-C1-4alkyl, hydroxy-C1-4 alkoxy, trifluoromethoxy, carboxy, —COOR5 where R5 is an ester group, —CONR6R7 or —NR6R7 where R6 and R7 are each hydrogen or C1-4 alkyl;    R2 is phenyl, napthyl or heteroaryl selected from thienyl, pyridyl, benzothienyl, quinolinyl, benzofuranyl or benzimidazolyl, wherein said phenyl, napthyl and heteroaryl groups are optionally substituted, or R2 is furanyl optionally substituted with C1-4 alkyl;    R3 is nitrile, carboxy, —COOR8 where R8 is an ester group, —CONR9R10 where R9 and R10 are each hydrogen or C1-4 alkyl or R11SO2 where R11 is C1-4 alkyl or optionally substituted phenyl;    R4 is —NR12R13, —NHCOR2, —N(COR12)2 or —N═CHOCH2R12 where R12 and R13 are each hydrogen or C1-4 alkyl optionally substituted with carboxy, or R4 is
    where X is C2-4 alkylene, or R4 is —NHSO2R14 where R14 is C1-4 alkyl or optionally substituted phenyl; and    n is 0–2.
U.S. Pat. No. 5,281,619 discloses naphthopyrans for therapy of diabetic complications:
wherein,    R1 is C1-4 alkoxy, OH or COOH;    R2 is optionally substituted phenyl;    R3 is nitrile, or R3 is carboxy or —COOR8 when R2 is phenyl substituted with 3-nitro or 3-trifluoromethyl and R8 is an ester group;    R4 is NR12R13, —NHCOR2, —N(COR2)2 or —N═CHOCH2R12, wherein R12 and R13 are each H or C1-4 alkyl; and    n is 0–2.
EP599514 discloses the preparation of pyranoquinoline derivatives as inhibitors of cell proliferation:
wherein R1 is optionally substituted phenyl or optionally substituted heteroaryl selected from thienyl, pyridyl, benzothienyl, quinolinyl, benzofuranyl or benzimidazolyl, or R1 is furanyl optionally substituted with C1-4 alkyl;    R2 is nitrile, carboxy, —CO2R4 wherein R4 is an ester group, —CON(R5)R6 where R5 and R6 are independently H or C1-4 alkyl, or R7SO2 where R7 is C1-4 alkyl or optionally substituted phenyl;    R3 is —NR8R9, —NHCOR8, —N(CO2R8)2, —N═CHOR8 where R8 and R9 are independently H or C1-4 alkyl, or —NHSO2R10 where R10 is C1-4 alkyl or optionally substituted phenyl, or
    where X is C2-4 alkylene; and    the ring P represents a pyridine fused to the benzopyran nucleus.
EP6 18206 discloses the preparation of naphthopyran and pyranoquinoline as immunosuppressants and cell proliferation inhibitors:
wherein,    A-B is CH2CH2 or CH═CH;    each R1 is independently halo, carboxy, trifluoromethyl, hydroxy, C1-4 alkyl, C1-4 alkoxy, C1-4 alkylthio, hydroxy-C1-4alkyl, hydroxy-C1-4alkoxy, nitrogen-containing heterocyclyl, nitro, trifluoromethoxy, —COOR5 where R5 is an ester group, —COR6, —CONR6R7 or —NR6R7 where R6 and R7 are each hydrogen or C1-4 alkyl;    R2 is phenyl, napthyl or heteroaryl selected from thienyl, pyridyl, benzothienyl, quinolinyl, benzofuranyl or benzimidazolyl, wherein said phenyl, napthyl and heteroaryl groups are optionally substituted, or R2 is furanyl optionally substituted with C1-4 alkyl;    R3 is nitrile, carboxy, —COOR8 where R8 is an ester group, —CONR9R10 where R9 and R10 are each hydrogen or C1-4 alkyl, or —SO2R11 where R11 is C1-4 alkyl or optionally substituted phenyl-C1-4 alkyl;    R4 is 1-pyrrolyl, 1-imidazolyl or 1-pyrazolyl, each of which is optionally substituted by one or two C1-4 alkyl, carboxyl, hydroxyl-C1-4 alkyl or —CHO groups, or R4 is 1-(1,2,4-triazolyl), 1-(1,3,4-triazolyl) or 2-(1,2,3-triazolyl), each of which is optionally substituted by a C1-4 alkyl or C1-4 perfluoroalkyl group, or R4 is 1-tetrazolyl optionally substituted by C1-4 alkyl;    X is a pyridine or a benzene ring; and    n is 0–2.
EP619314 discloses the preparation of 4-phenyl-4H-naphtho(2,1-b)pyran derivatives:
wherein,    R1 and R2 are independently halo, trifluoromethyl, C1–C4 alkoxy, hydroxy, nitro, C1–C4 alkyl, C1–C4 alkylthio, hydroxy-C1–C4 alkyl, hydroxy-C1–C4alkoxy, trifluoromethoxy, carboxy, —COOR8 where R8 is an ester group, —COR9, —CONR9R10 or —NR9R10 where R9 and R10 are each hydrogen or C1–C4 alkyl;    R3 is nitrile, carboxy or —CO2R11 wherein R11 is an ester group;    R4 is —NR12R13, —NR12COR13, —N(COR12)2 or —N═CHOCH2R12 where R12 and R13 are each hydrogen or C1-4 alkyl, or R4 is
    where X is C2–C4 alkylene, or R4 is optionally substituted 1-pyrrolyl; and    m and n are each independently 0–2.The compounds are said to be useful for the treatment of restenosis, immune disease, and diabetic complications.
Smith, et al., (Bioorg. Med. Chem. Lett. 5:2783–2788 (1995)) reported the anti-rheumatic potential of a series of 2,4-di-substituted-4H-naphtho[1,2-b]pyran-3-carbonitriles. They reported that 4-(3-nitrophenyl)-2-(N-succinimido)-4H-naphtho[1,2-b]pyran-3-carbonitrile has proved to be acid stable and still retains biological activity:

Birch, et al., (Diabetes 45:642–650 (1996)) reported that LY290181, an inhibitor of diabetes-induced vascular dysfunction, blocks protein kinase C-stimulated transcriptional activation through inhibition of transcription factor binding to a phorbol response element:

Panda, et al., (J. Biol. Chem. 272: 7681–7687 (1997)) reported the suppression of microtubule dynamics by LY290181, which might be the potential mechanism for its antiproliferative action.
Wood, et al., (Mol. Pharmacol. 52: 437–444 (1997)) reported that LY290181 inhibited mitosis and microtubule function through direct tubulin binding.
PCT published patent application WO9824427 disclosed antimicrotubule compositions and methods for treating or preventing inflammatory diseases. LY290181 was listed as an antimicrotubule agent.